Project Summary This application is set to define a novel mechanism underlying the control of gut microbiota-immune checkpoint interactions by the ubiquitin ligase RNF5, and the implications of such regulation to melanoma development and response to therapy. Despite the most exciting and significant advances made in clinical management of melanoma via the immune-checkpoint based clinical trials, mechanisms underlying their control, the susceptibility to distinct therapies (or not), are among the fundamental questions that remain unsolved. Here, we provide data to support a model whereby immune checkpoints are regulated by gut microbiome, which is defined by the ubiquitin ligase RNF5. Our discovery of impaired growth of Braf/Pten mutant melanoma in syngeneic Rnf5?/? mice, compared with Rnfwt littermates, was linked with enhanced infiltration of tumor- infiltrating lymphocytes (TILs) (CD4 and CD8 positive), macrophages and dendritic cells in the tumors that developed significantly slower in the Rnf5?/? mice. Strikingly, co-housing the Rnf5?/? and Rnfwt animals or antibiotic ablation of the gut microbiota resulted in loss of the above phenotypes?tumor growth was no longer attenuated and immune checkpoint-based phenotypes were largely lost. Assessment of the gut microbiome revealed a distinct subset of bacterial species, which distinguish Rnf5?/? mice from their WT littermates (all ?pure? in-house maintained C57BL/6 strain). Notably, common to the distinct RNF5-microbiome are bacterial species that generate select subset of short chain fatty acids. These observations provide the foundation for our hypothesis that RNF5 controls the intestinal microbiota that affects immune checkpoint mechanisms, which in turn impacts melanoma development. Our proposed studies will (i) define RNF5 effect on the gut microbiome?tumor interactions, (ii) identify microbiome-dependent changes in the regulation of tumor immune checkpoint control by RNF5, and (iii) establish the physiological significance and impact of RNF5 control of microbiome and immune checkpoint on melanomagenesis in different mouse strains, age, and under select combination therapies. Our highly integrated studies will define the fundamental mechanisms that underlie the regulation of both gut microbiome and immune checkpoints, thereby providing new insights into therapeutic modalities for melanoma and other cancers.